Activation of a PP2A-like phosphatase and dephosphorylation of tau protein characterize onset of the execution phase of apoptosis.

The execution phase is an evolutionarily conserved stage of apoptosis that occurs with remarkable temporal and morphological uniformity in most if not all cell types regardless of the condition used to induce death. Characteristic features of apoptosis such as membrane blebbing, DNA fragmentation, chromatin condensation, and cell shrinkage occur during the execution phase; therefore, there is considerable interest in defining biochemical changes and signaling events early in the execution phase. Since onset of the execution phase is asynchronous across a population with only a small fraction of cells in this stage at any given time, characterizing underlying biochemical changes is difficult. An additional complication is recent evidence suggesting that the execution phase occurs after cells commit to die; thus, agents that modulate events in the execution phase may alter the morphological progression of apoptosis but will not affect the time-course of death. In the present study, we use a single cell approach to study and temporally order biochemical and cytoskeletal events that occur specifically in the execution phase. Microtubules de-acetylate and disassemble as terminally differentiated PC12 cells enter the execution phase following removal of nerve growth factor. Using phosphorylation sensitive antibodies to tau, we show that this microtubule-stabilizing protein becomes dephosphorylated near the onset of the execution phase. Low concentrations of okadaic acid inhibit dephosphorylation suggesting a PP2A-like phosphatase is responsible. Transfecting (tau) into CHO cells to act as a 'reporter' protein shows a similar dephosphorylation of (tau) by a PP2A-like phosphatase during the execution phase following induction of apoptosis with UV irradiation. Therefore, activation of PP2A phosphatase occurs at the onset of the execution phase in two very different cell types following different initiators of apoptosis which is consistent with activation of PP2A phosphatase being a common feature of the execution phase of apoptosis. Experiments using either taxol to inhibit microtubule disassembly or okadaic acid to inhibit tau dephosphorylation suggest that microtubule disassembly is necessary for tau dephosphorylation to occur. Thus, we propose that an early step in the execution phase (soon after a cell commits to die) is microtubule disassembly which frees or activates PP2A to dephosphorylate tau as well as other substrates.